TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

Cu(II) Coordination Polymers Containing Mixed Ligands with Different Flexibilities: Structural Diversity and Iodine Adsorption.

Reactions of N,N'-bis(3-methylpyridyl)oxalamide (L1), N,N'-bis(3-methylpyridyl)adipoamide (L2) and N,N'-bis(3-methylpyridyl)sebacoamide (L3) with tricarboxylic acids and Cu(II) salts afforded {[Cu(L1)(1,3,5-HBTC)]·H2O}n (1,3,5-H3BTC = 1,3,5-benzenetricarboxylic acid), 1, {[Cu1.5(L2)1.5(1,3,5-BTC)(H2O)2]·6.5H2O}n, 2, [Cu(L2)0.5(1,3,5-HBTB)]n (1,3,5-H3BTB = 1,3,5-tri(4-carboxyphenyl)benzene), 3, [Cu4(L3)(OH)2(1,3,5-BTC)2]n, 4, {[Cu3(L3)2(1,3,5-BTB)2]·2.5MeOH·2H2O}n, 5, and {[Cu3(L3)2(1,3,5-BTB)2 ]·DMF·2H2O}n, 6, which have been structurally characterized by using single crystal X-ray crystallography. Complexes 1-4 form a 2D layer with the {44.62}-sql topology, a 2D layer with the (4.62)2(42.62.82)-bex topology, a three-fold interpenetrated 3D net with the (412·63)-pcu topology and a 3D framework with the (410·632·83)(42·6)2(43·63) topology, respectively, whereas 5 and 6 are 3D frameworks with the (63)2(64·82)(68·85·102) topology. Complex 5 shows a better iodine adsorption factor of 290.0 mg g-1 at 60 °C for 360 min than the other ones, revealing that the flexibility of the spacer ligand governs the structural diversity and the adsorption capacity.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target.

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.

Suspension Trapping-Based Sample Preparation Workflow for In-Depth Plant Phosphoproteomics.

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Despite the widespread use of protein precipitation for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro-column with a Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis (Arabidopsis thaliana) phosphoproteome by more than 30%, with improved number of multiply phosphorylated peptides, quantification accuracy, and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus discovered that a significant proportion of the phosphopeptides induced by ABA are multiply phosphorylated peptides, indicating their importance in early ABA signaling and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.

Far-UV absorption spectra of SiH2 and dibridged Si2H2 isolated in solid argon.

We employ electron bombardment during the deposition of an Ar matrix containing a small proportion of SiH4 to generate various silicon hydrides. Subsequently, the irradiation of a matrix sample at 365 nm decomposes SiH2 and dibridged Si2H2 in solid Ar, which we identify through infrared spectroscopy. We further recorded the corresponding ultraviolet absorption spectra at each experimental stage. An intense band observed in the range of 170-203 nm is largely destroyed upon 365-nm photolysis, which is assigned to the C1B2 â† X1A1 transition of SiH2. Moreover, a moderate band observed in the region of 217-236 nm is reduced slightly, which is assigned to the 31B2 â† X1A1 transition of dibridged Si2H2. These assignments are made based on the observed photolytic behavior, and the prediction of the vertical excitation energies with the corresponding oscillator strengths by using time-dependent density functional theory and equation-of-motion coupled cluster theory.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1ß is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.

Development of Furanopyrimidine-Based Orally Active Third-Generation EGFR Inhibitors for the Treatment of Non-Small Cell Lung Cancer.

The development of orally bioavailable, furanopyrimidine-based double-mutant (L858R/T790M) EGFR inhibitors is described. First, selectivity for mutant EGFR was accomplished by replacing the (S)-2-phenylglycinol moiety of 12 with either an ethanol or an alkyl substituent. Then, the cellular potency and physicochemical properties were optimized through insights from molecular modeling studies by implanting various solubilizing groups in phenyl rings A and B. Optimized lead 52 shows 8-fold selective inhibition of H1975 (EGFRL858R/T790M overexpressing) cancer cells over A431 (EGFRWT overexpressing) cancer cells; western blot analysis further confirmed EGFR mutant-selective target modulation inside the cancer cells by 52. Notably, 52 displayed in vivo antitumor effects in two different mouse xenograft models (BaF3 transfected with mutant EGFR and H1975 tumors) with TGI = 74.9 and 97.5% after oral administration (F = 27%), respectively. With an extraordinary kinome selectivity (S(10) score of 0.017), 52 undergoes detailed preclinical development.

Formation of Para-H2O by Vacuum-UV Photolysis of O2 in Solid Hydrogen: Implication for Astrochemistry.

The observation that the ortho to para ratio (OPR) of interstellar H2O is smaller than 3 is an important yet unresolved subject in astronomy. We irradiated O2 embedded in solid H2 at 3 K with vacuum-ultraviolet (VUV) light and observed IR lines associated with para-H2O (denoted as pH2O) and nonrotating H2O-(oH2)n (where oH2 denotes ortho-H2) but no lines associated with ortho-H2O (denoted as oH2O). After maintaining the matrix in darkness for ∼30 h, the amount of pH2O decreased, accompanied by an increase in H2O-(oH2)n via diffusion of oH2. After that, the continuous nuclear-spin conversion from oH2 to para-H2 (denoted as pH2) in solid H2 over time resulted in the conversion of nonrotating H2O-(oH2)n to rotating pH2O in solid pH2. The observation of the formation and conversion of pH2O in our experiment suggests a plausible route in which VUV irradiation of O2 and H2 adsorbed on grain surfaces might be responsible for the smaller OPR of interstellar H2O.

Selective and antagonist-dependent µ-opioid receptor activation by the combination of 2-{[2-(6-chloro-3,4-dihydro-1(2H)-quinolinyl)-2-oxoethyl]sulfanyl}-5-phenyl-4,6-(1H,5H)-pyrimidinedione and naloxone/naltrexone.

We identified, via high-throughput screening using a FLIPR® calcium assay, compound 1, which incorporated a dihydroquinolinyl-2-oxoethylsulfanyl-(1H,5H)-pyrimidinedione core and activated the µ-opioid receptor (MOR) in the presence of naloxone or naltrexone. A structure-activity relationship study of the analogs of 1 led to the design of compound 21, which activated MOR in the presence of naloxone with an EC50 of 3.3 ± 0.2 µM. MOR activation by the compound 21-antagonist pair was antagonist-dependent. Compound 21 did not affect the potency of the orthosteric agonist, morphine, toward MOR, indicating that it affected the function of MOR antagonists rather than that of the agonists. Computer modeling of the compound 21-MOR-naloxone complex revealed major interactions between compound 21 and MOR, including hydrogen bonding with Ser196, π-π stacking with Tyr149, and sulfur-aromatic interaction with Trp192. This study may pave the way for developing agents capable of safe and effective MOR modulation.

Tumor suppressor BAP1 nuclear import is governed by transportin-1.

Subcellular localization of the deubiquitinating enzyme BAP1 is deterministic for its tumor suppressor activity. While the monoubiquitination of BAP1 by an atypical E2/E3-conjugated enzyme UBE2O and BAP1 auto-deubiquitination are known to regulate its nuclear localization, the molecular mechanism by which BAP1 is imported into the nucleus has remained elusive. Here, we demonstrated that transportin-1 (TNPO1, also known as Karyopherin ß2 or Kapß2) targets an atypical C-terminal proline-tyrosine nuclear localization signal (PY-NLS) motif of BAP1 and serves as the primary nuclear transporter of BAP1 to achieve its nuclear import. TNPO1 binding dissociates dimeric BAP1 and sequesters the monoubiquitination sites flanking the PY-NLS of BAP1 to counteract the function of UBE2O that retains BAP1 in the cytosol. Our findings shed light on how TNPO1 regulates the nuclear import, self-association, and monoubiquitination of BAP1 pertinent to oncogenesis.

IR absorption spectra of aniline cation, anilino radical, and phenylnitrene isolated in solid argon.

Electron bombardment of aniline (PhNH2) in an Ar matrix mainly generated the aniline cation (PhNH2+), anilino (PhNH) and phenyl (Ph) radicals, and phenylnitrene (PhN). Further irradiation of the electron-bombarded matrix sample at 365 nm depleted PhNH2+ and PhN, and resulted in the formation of PhNH2, PhNH, and Ph. In separate experiments, irradiation of the PhNH2/Ar matrix samples at 265 or 160 nm mainly generated PhNH and Ph radicals, but without the formation of PhNH2+ and PhN. According to the observed photochemical behaviors, quantum-chemically predicted harmonic vibrational wavenumbers of each species, and the information reported in previous photodissociation studies, we unambiguously characterized the IR features of the aromatic species. The information of the vibrational fundamentals of PhNH is new and the formation mechanism is discussed.

K48/K63-linked polyubiquitination of ATG9A by TRAF6 E3 ligase regulates oxidative stress-induced autophagy.

Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. Here, we show that TRAF6 E3 ubiquitin ligase and A20 deubiquitinase coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The ROS-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of VPS34-UVRAG complex, thereby stimulating autophagy. Notably, expression of the ATG9A ubiquitination mutants impairs ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreases IRF-3 phosphorylation in LPS-stimulated macrophages. Our findings provide important insights into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.

Electrochemically-functionalized CNT/ABTS nanozyme enabling sensitive and selective voltammetric detection of microalbuminuria.

Developing a cost-effective and reliable sensing platform for the routine screening of microalbuminuria and early diagnosis of chronic kidney disease is of great importance. Herein, we report on the high specificity and electrocatalytic activity of CNT/ABTS(CV) nanozyme, derived from the electrochemical functionalization of the carbon nanotube/2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) nanocomposite CNT/ABTS), towards electrochemical oxidation of human serum albumin (HSA) in urine. The potential cycling of CNT/ABTS electrode resulted in the surface functionalization with imine and carbonyl functional groups, which enhances electrode's affinity towards the electroactive tyrosine and tryptophan amino acids and thus facilitates the adsorption and subsequent electrochemical oxidation of HSA. The developed CNT/ABTS(CV) nanozyme modified electrode showed promising sensing characteristics for the point-of-care diagnosis of microalbuminuria without any sample pretreatment and inclusion of additional label or redox probes during measurement, including a practical sensing range up to 1.50 µM, a low detection limit of 60 nM, good reusability (>5 cycles), good long-term stability (>28 days), and high accuracy (error <15%) for the detection of HSA in urine. This work opens a new avenue for the detection and quantification of proteins in physiological conditions.

Far-UV spectroscopy of mono- and multilayer hexagonal boron nitrides.

Hexagonal boron nitrides (hBNs) have a very high luminescence efficiency and are promising materials for deep-UV emitters. Although intense deep-UV emissions have been recorded in various forms of hBN excited by photons or energetic electrons, information on the electronic structure of the conduction band has been derived mainly from theoretical works. Therefore, there is a lack of high-resolution absorption data in the far-UV region. In this study, the far-UV absorption spectra of chemical-vapor-deposition-grown mono- and multilayer hBNs were recorded at 10 and 298 K. In addition to the previously reported band at 6.10 eV, two absorption bands at 6.82 and 8.86 eV were observed for the first time in thin-film hBN. Furthermore, excitation of the hBN thin film samples with 6.89-eV photons revealed intense emission peaks at 6.10 (mono) and 5.98 (multi) eV with a bandwidth of ∼0.7 eV. Comparing the absorption and photoluminescence data, we believe that both direct and indirect transitions occur in the radiative processes.

Side-by-Side Comparison of Human Perception and Performance Using Augmented, Hybrid, and Virtual Reality.

Alternative reality (XR) technologies, including physical, augmented, hybrid, and virtual reality, offer ways for engineered spaces to be evaluated. Traditionally, practitioners (such as those designing spacecraft habitats) have relied on physical mockups to perform such design evaluations, but digital XR technologies present several streamlining advantages over their physical counterparts. These digital environments vary in their level of virtuality, and consequently have different effects on human perception and performance, with respect to a completely physical mockup environment. To date, very little has been done to characterize and quantify such differences in human perception and performance across XR environments of equal fidelity for the same end application. Here, we show that perception and performance in the virtual reality environment most closely mirror those in the physical reality environment, as measured through volumetric assessment and functional task experiments. These experiments required subjects to judge the dimensions of 3D objects and perform operational tasks presented via checklists. Our results highlight the potential for virtual reality systems to accelerate the iterative design of engineered spaces relative to the use of physical mockups, while preserving the human perception and performance characteristics of a completely physical environment. These findings also elucidate specific advantages and disadvantages to specific digital XR technologies with respect to one another and the physical reality baseline. Practitioners may inform their selection of an XR modality for their specific end application based on this comparative analysis, as it contextualizes the niche for each technology in the realm of iterative design for engineered spaces.

Colloidal Assemblies Composed of Polymeric Micellar/Emulsified Systems Integrate Cancer Therapy Combining a Tumor-Associated Antigen Vaccine and Chemotherapeutic Regimens.

Integrative medicine comprising a tumor-associated antigen vaccine and chemotherapeutic regimens has provided new insights into cancer therapy. In this study, the AB-type diblock copolymers poly(ethylene glycol)-polylactide (PEG-PLA) were subjected to the dispersion of poorly water-soluble molecules in aqueous solutions. The physicochemical behavior of the chemotherapeutic agent DBPR114 in the PEG-PLA-polymeric aqueous solution was investigated by dynamic light scattering (DLS) technology. In vitro cell culture indicated that replacing the organic solvent DMSO with PEG-PLA polymeric micelles could maintain the anti-proliferative effect of DBPR114 on leukemia cell lines. A murine tumor-associated antigen vaccine model was established in tumor-bearing mice to determine the effectiveness of these formulas in inducing tumor regression. The results demonstrated that the therapeutic treatments effectively reinforced each other via co-delivery of antitumor drug/antigen agents to synergistically integrate the efficacy of cancer therapy. Our findings support the potential use of polymeric micellar systems for aqueous solubilization and expansion of antitumor activity intrinsic to DBPR114 and tumor-associated antigen therapy.

Novel Stable Capacitive Electrocardiogram Measurement System.

This study presents a noncontact electrocardiogram (ECG) measurement system to replace conventional ECG electrode pads during ECG measurement. The proposed noncontact electrode design comprises a surface guard ring, the optimal input resistance, a ground guard ring, and an optimal voltage divider feedback. The surface and ground guard rings are used to reduce environmental noise. The optimal input resistor mitigates distortion caused by the input bias current, and the optimal voltage divider feedback increases the gain. Simulated gain analysis was subsequently performed to determine the most suitable parameters for the design, and the system was combined with a capacitive driven right leg circuit to reduce common-mode interference. The present study simulated actual environments in which interference is present in capacitive ECG signal measurement. Both in the case of environmental interference and motion artifact interference, relative to capacitive ECG electrodes, the proposed electrodes measured ECG signals with greater stability. In terms of R-R intervals, the measured ECG signals exhibited a 98.6% similarity to ECGs measured using contact ECG systems. The proposed noncontact ECG measurement system based on capacitive sensing is applicable for use in everyday life.

Use of seroprevalence to guide dengue vaccination plans for older adults in a dengue non-endemic country.

A shift in dengue cases toward the adult population, accompanied by an increased risk of severe cases of dengue in the elderly, has created an important emerging issue in the past decade. To understand the level of past DENV infection among older adults after a large dengue outbreak occurred in southern Taiwan in 2015, we screened 1498 and 2603 serum samples from healthy residents aged ≥ 40 years in Kaohsiung City and Tainan City, respectively, to assess the seroprevalence of anti-DENV IgG in 2016. Seropositive samples were verified to exclude cross-reaction from Japanese encephalitis virus (JEV), using DENV/JEV-NS1 indirect IgG ELISA. We further identified viral serotypes and secondary DENV infections among positive samples in the two cities. The overall age-standardized seroprevalence of DENV-IgG among participants was 25.77% in Kaohsiung and 11.40% in Tainan, and the seroprevalence was significantly higher in older age groups of both cities. Although the percentages of secondary DENV infection in Kaohsiung and Tainan were very similar (43.09% and 44.76%, respectively), DENV-1 and DENV-2 spanned a wider age range in Kaohsiung, whereas DENV-2 was dominant in Tainan. As very few studies have obtained the serostatus of DENV infection in older adults and the elderly, this study highlights the need for further investigation into antibody status, as well as the safety and efficacy of dengue vaccination in these older populations.

VPS34 K29/K48 branched ubiquitination governed by UBE3C and TRABID regulates autophagy, proteostasis and liver metabolism.

The ubiquitin-proteasome system (UPS) and autophagy are two major quality control processes whose impairment is linked to a wide variety of diseases. The coordination between UPS and autophagy remains incompletely understood. Here, we show that ubiquitin ligase UBE3C and deubiquitinating enzyme TRABID reciprocally regulate K29/K48-branched ubiquitination of VPS34. We find that this ubiquitination enhances the binding of VPS34 to proteasomes for degradation, thereby suppressing autophagosome formation and maturation. Under ER and proteotoxic stresses, UBE3C recruitment to phagophores is compromised with a concomitant increase of its association with proteasomes. This switch attenuates the action of UBE3C on VPS34, thereby elevating autophagy activity to facilitate proteostasis, ER quality control and cell survival. Specifically in the liver, we show that TRABID-mediated VPS34 stabilization is critical for lipid metabolism and is downregulated during the pathogenesis of steatosis. This study identifies a ubiquitination type on VPS34 and elucidates its cellular fate and physiological functions in proteostasis and liver metabolism.